In many nerve cells, transient increases in intracellular free calcium concentrations (Caj) are caused primarily by influx through voltage- dependent Ca channels. Second messengers like inositol triphosphate (InsP3) also have the ability to increase Caj) through release from intracellular stores, or gating of calcium channels. The long-term goal of this research is to investigate mechanisms by which second messengers such as Ca2+ or InsP3 modulate the excitability of nerve cells by controlling their membrane permeability. We have developed suitable technologies: 1) to measure single-channel activities ii) to simultaneously measure changes in intracellular Ca and membrane currents; iii) to pressure-inject pharmacological agents to investigate putative pathways involved in neuronal excitability. There are three specific aims of the proposed research. 1. We will test the hypothesis that InsP3 directly activates ion channels in the plasma membrane of isolated nerve cells by recording single-channels gated by InsP3 in excised patches. We will determine properties of the excise patch by: 1) puffing different concentrations of InsP3. ii) comparing InsP3 or InsP4 on the same patch. iii) puffing InsP3 with different concentration of Ca2+. iv) puffing InsP3 with heparin. 2. We will determine whether the Cai increase resulting from Ca influx is potentiated by release of calcium form intracellular stores by injecting heparin or applying thapsigargin, agents that inhibit the action of InsP3 or induce depletion of intracellular stores. Intracellular calcium will be measured with Ca-sensitive electrodes. 3. We will directly test the hypothesis that the inability of InsP3 to release additional Ca2+ from internal stores following a prior mobilization of Ca2+ is due to a lingering elevation of Cai and the presence of an additional factor present only in certain areas of the cell. We will determine the threshold of desensitization of InsP3 injections by injecting increasing amounts of calcium. We will simultaneously measure Cai with aequorin and Ca-sensitive electrodes. The combination of these electrophysiological and pharmacological techniques should prove useful in gathering new and important information about nerve cell function. In particular, the studies on the long lasting afterdischarge in nerve cells, may contribute to a better understanding of the long lasting hyperexcitability of human brain cells in epilepsy.